Xfe96

Real-time oxygen consumption rates (OCR) were determined using the Seahorse Extracellular Flux analyzer (XF96) (Agilent). Adrenocortical cancer cells (H295R, SW13, MUC-1) and H295R clones (shCTR, shERRα−/−, ERRα+/+) were seeded into XF96-well cell culture plates (Seahorse Bioscience: North Billerica, MA, USA) and incubated overnight at 37 °C in a 5% CO₂ humidified atmosphere. After 48 h, cells were treated with XCT790 (1, 5, 10 μM) for 18 h. At the end of treatment, cells were washed in warm XF assay media supplemented with 10 mM glucose, 1 mM Pyruvate, 2 mM L-glutamine and adjusted at pH 7.4. Cells were then maintained for 1 h in 175 μL/well of XF assay media at 37 °C, in a non-CO₂ incubator. During the cell incubation time, 25 μL of a solution of XF assay media containing 10 μM oligomycin, 9 μM FCCP, 10 μM rotenone, 10 μM antimycin A were loaded into the injection ports of the XFe-96 sensor cartridge. The dataset was analyzed by XFe-96 software (Agilent).

The extracellular acidification rate in real time (ECAR) was determined using the Seahorse Extracellular Flux Analyzer (XF96) (Agilent). Adrenocortical cancer cells (H295R, SW13, MUC-1) and H295R clones (shCTR, shERRα−/−, ERRα+/+) were seeded into XF96-well cell culture plates (Seahorse Bioscience, MA, USA), and incubated overnight at 37 °C in a 5% CO₂ humidified atmosphere. After 48 h, cells were treated with XCT790 (1, 5, 10 μM) for 18 h. At the end of treatment, cells were washed in a specific buffer (XF medium, pH 7.4) for the determination of metabolic flows added with 2 mM of L-glutamine. The cells were then maintained for 1 h in 175 μL of XF medium at 37 °C, in an incubator without CO₂. During the incubation time, the XF buffer solution (25 µL) containing glucose (10 mM) oligomycin (1μM), 2-deoxy-D-glucose (50 mM) was added into the injection ports. ECAR measurements were normalized to protein content within the individual wells. The dataset was analyzed by XFe-96 software (Agilent).

Multiparameter metabolic analysis of MDA-MB-231 cells was performed in an extracellular flux analyzer XFe96 (Seahorse Bioscience, USA) as described^{95 (link)}. Details are provided in the supplementary document.

Briefly, following the dosing period, media was switched to unbuffered XF basal medium (175 μl, pH 7.4) supplemented with glucose (25 mM), l-glutamine (2 mM) and sodium pyruvate (1 mM) before incubating in a CO₂ free incubator (37 °C). Oxygen consumption was measured by Seahorse XFe96 according to the previously described protocol (Kamalian et al., 2018 (link)).
The assessment of individual mitochondrial complex activity was preformed using the in-situ mito substrate assay in permeabilised cells using Seahorse Technology as previously described (Ball et al., 2016 (link)).

Mitostress test analysis was performed in a Seahorse XFe96 instrument. The equal numbers of cells (10,000 cells/well) were seeded in a XFe96 culture microplate. They were cultured in standard media in 5% CO₂ at 37 °C and treated with 10 µM ONC201 for 24 h. Mitostress analysis protocol was followed according to the manufacturers guidelines (Agilent). In brief, on the day of experiment prior to the run, culture media from the culture microplate was replaced with seahorse XF assay media (Agilent, California, USA) supplemented with 1 mM Pyruvate, 2 mM Glutamine and 10 mM glucose (bicarbonate free). Oxygen consumption rate (OCR) were measured at baseline, after addition of oligomycin 2 μM, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) 2 μM and rotenone 0.5 μM. Data were normalised by cell numbers after the treatment. The metabolic and respiratory parameters were analysed in the wave software.